Upon completion of the human genome project, genotyping of individuals will become possible and important to trace biological reactions of each individual. This is one of the conceptions for health care in the future. Molecular medicine is expected to evolve for each individual based on his or her genetic identity. Chip-based nucleic acid sequence analysis is a powerful tool for re-sequencing gene to identify single nucleotide polymorphisms (SNP), which is believed to be responsible for individual identity. Spacially addressed primer array offers a cheap and effective method for SNP detection. However, to justify the signal of each spot in the entire array and to analyze the complete data is very tedious and requires decent software. Herein, I describe a novel format of arrayed primer extension, which simplifies the data analysis and leads to straightforward answer.
The current art of spotting microarray on a chip has the oligonucleotides laid on a chip sequentially from one end (3xe2x80x2 or 5xe2x80x2) to the other end (5xe2x80x2 or 3xe2x80x2) of the target. As shown in FIG. 1, the primers add one base difference as they proceed from one end to the other end. A complete hybridization map to the arrayed primers reveals the sequence of the gene in question.
FIG. 2 shows a fluorescent image of a conventional oligonucleotide array which has been hybridized with a sample nucleic acid and subjected to a primer extension reaction. The four terminators are labeled with four fluorescent dyes. Each terminator that has been incorporated onto the oligonucleotide primer may be identified on the image by color. The image of the dye terminator labeled spots was obtained using an Avalanche microscanner (Molecular Dynamics, Sunnyvale, Calif.). As depicted in FIG. 2, it is difficult to ascertain the presence of mutations in samples using such sequentially placed oligonucleotide primer arrays without the aid of complex software.
While nucleotide arrays present many advantages in the analysis of nucleic acid sequences, improvements in the techniques incorporating such arrays are still possible.
A method for detecting a mutation in a target nucleic acid sequence that comprises: attaching oligonucleotide primers to a substrate, wherein the oligonucleotide primers have a sequence that is complementary to the target nucleic acid sequence, and wherein the oligonucleotide primers are grouped according to the identity of the first base which would be expected to be added to the primer through the process of primer extension; hybridizing to the oligonucleotide primers a sample nucleic acid sequence which possibly contains a mutation; extending each oligonucleotide primer by one base using a reaction mixture comprising labeled ddNTPs and enzyme; and detecting a mutation in the sample nucleic acid sequence by detecting the presence of a labeled ddNTP which does not correspond to the identity of the base expected to be added to the primer through the process of primer extension. The sample nucleic acid sequence may be mRNA or DNA. The labeled ddNTPs may be labeled with a fluorescent dye, a chemiluminescent reagent, a radioactive label, a redox tag, or an electrically conductive tag.
The instant invention also pertains to a nucleic acid array comprising a substrate containing oligonucleotide primers having a sequence complementary to a target nucleic acid sequence, wherein the oligonucleotide primers are grouped according to the identity of the first base which would be expected to be added to the primer through the process of primer extension after hybridizing with a sample nucleic acid sequence.
The instant invention also pertains to a kit for detecting genetic mutations comprising the nucleic acid array as described above.
The instant invention also pertains to a method for detecting a mutation in a target nucleic acid sequence that comprises: identifying the base expected to be added to a primer located at a particular coordinate on an oligonucleotide array as expected from the target nucleic acid sequence; identifying the base actually added to the primer located at the particular coordinate on the oligonucleotide array through the process of primer extension; comparing the base actually added to the primer at the particular coordinate with the base expected to be added to the primer at the particular coordinate; and reporting those instances where the bases are not the same, in order to identify a mutation.